The studies proposed in this grant application represent a detailed and focused effort to formally investigate the genetic and functional impact of a newly identified polymorphism (P227A) in human CD40, a protein with a critical role in regulating immune system functions. The P227A polymorphism results in a nonsynonomous substitution of alanine (A) for proline (P) at position 227 in the intracellular protein sequence of CD40 near the TRAF6 recognition site. Our preliminary data demonstrate that: 1) P227A is a naturally occurring polymorphism with an estimated allele frequency of approximately 1.5% in Caucasians, 2) in SLE families that carry P227A the polymorphism is preferentially transmitted to SLE affected individuals, 3) 293T cells transfected with P227A demonstrate constitutive activation of NF-DB, AP-1 and Ca2+/cAMP response element (CRE) transcription factors, 4) CD40 P227A results in TRAF2 binding constitutively to CD40 in the absence of CD40 ligand (CD40L). In order to expand our understanding of the influence of P227A in the predisposition to autoimmune disease, a thorough analysis of the genetic prevalence of the P227A in various normal and autoimmune disease populations and the consequences of P227A in normal CD40 dependent immune mechanisms is needed. To do this we propose three specific aims, each designed to test important hypotheses regarding P227A genetics and function. In the first aim we will investigate the prevalence of P227A in defined control populations of varying ethnicity, individuals and families with SLE and other autoimmune diseases and investigate the prevalence of clinical and serologic signs of immune hyperactivity in autoimmune and nonautoimmune P227A carriers. In aim 2 we will test the P227A "gain-of-function" hypothesis by exploring the influence of P227A on CD40 dependent immune functions in freshly isolated peripheral blood mononuclear cells (PBMC) from carriers. Further studies in aim 2 will investigate specific mechanisms by which P227A could predispose to autoimmune disease by evaluating the effect of P227A on the maturation and function of circulating dendritic cells in the context of Toll-like receptor (TLR) signaling. Finally, in aim 3 we propose to develop an important and useful model system for further studies of this polymorphism, a chimeric CD40 knock-in mouse that expresses the murine ligand-binding domain coupled with the human intracellular CD40 WT or P227A domain.